Cell-permeable peptide inhibitors of the jnk signal transduction pathway

ABSTRACT

The invention provides cell-permeable peptides that bind to JNK protein and inhibit JNK-mediated effects in JNK-expressing cells.

FIELD OF THE INVENTION

This invention relates generally to protein kinase inhibitors and morespecifically to inhibitors of the protein kinase c-Jun amino terminalkinase.

BACKGROUND OF THE INVENTION

The c-Jun amino terminal kinase (JNK) is a member of thestress-activated group of mitogen-activated protein (MAP) kinases. Thesekinases have been implicated in the control of cell growth anddifferentiation, and, more generally, in the response of cells toenvironmental stimuli. The JNK signal transduction pathway is activatedin response to environmental stress and by the engagement of severalclasses of cell surface receptors. These receptors can include cytokinereceptors, serpentine receptors, and receptor tyrosine kinases. Inmammalian cells, JNK has been implicated in such biological processes asoncogenic transformation and in mediating adaptive responses toenvironmental stress. JNK has also been associated with modulatingimmune responses, including maturation and differentiation of immunecells, as well effecting programmed cell death in cells identified fordestruction by the immune system.

SUMMARY OF THE INVENTION

The present invention is based in part on the discovery of peptides thatare effective inhibitors of JNK proteins. The peptides, referred toherein as JNK peptide inhibitors, decrease the downstreamcell-proliferative effects of c-Jun amino terminal kinase (JNK).

Accordingly, the invention includes novel JNK inhibitor peptides, aswell as chimeric peptides which include a JNK peptide inhibitor linked atrafficking peptide that can be used to direct a peptide on which it ispresent do a desired cellular location. The trafficking sequence can beused to direct transport of the peptide across the plasma membrane.Alternatively, or in addition, the trafficking peptide can be used todirect the peptide to desired intracellular location, such as thenucleus.

The JNK inhibitor peptides can be present as polymers of L-amino acids.Alternatively, the peptides can be present as polymers of D-amino acids.

Also included in the invention are pharmaceutical compositions thatinclude the JNK-binding peptides, as well as antibodies thatspecifically recognize the JNK-binding peptides.

The invention also includes a method of inhibiting expression of a JNKkinase in a cell.

In another aspect, the invention includes a method of treating apathophysiology associated with activation of JNK in a cell or cells.For example, the target cells may be, e.g., cultured animal cells, humancells or micro-organisms. Delivery can be carried out in vivo byadministering the chimeric peptide to an individual in whom it is to beused for diagnostic, preventative or therapeutic purposes. The targetcells may be in vivo cells, i.e., cells composing the organs or tissuesof living animals or humans, or microorganisms found in living animalsor humans.

Among the advantages provided by the invention is that the JNK inhibitorpeptides are small, and can be produced readily in bulk quantities andin high purity. The inhibitor peptides are also resistant tointracellular degradation, and are weakly immunogenic. Accordingly, thepeptides are well suited for in vitro and in vivo applications in whichinhibition of JNK-expression is desired.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C are diagrams showing alignments of conserved JBD domainregions in the indicated transcription factors.

FIG. 2 is a diagram showing alignments of generic TAT-IB fusionpeptides.

FIG. 3 is a histogram depicting inhibition of n-cell death by theminimal 23 amino acid long JBD domain of IB1 compared to the full 280amino acid JBD domain.

FIG. 4 is an illustration demonstrating the effects of TAT, TAT-IB1 andTAT-IB2 peptides on phosphorylation of recombinant JNKs. Panel A showsinhibition of c-Jun, ATF2 and Elk1 phosphorylation by recombinant JNKsin vitro. Panel B shows dose response experiments similar to Panel A.

FIG. 5 is a histogram depicting L-TAT-IB inhibition of phosphorylationby recombinant JNKs. Panel A shows L-TAT-IB inhibition of c-Jun, ATF2and Elk1 phosphorylation by recombinant JNKs in vitro in the presence ofMKK4. Panel B shows similar dose response experiments with MKK7.

FIG. 6 is an illustration demonstrating the inhibition of c-Junphosphorylation by activated JNKs.

FIG. 7 is a histogram depicting short term inhibition of IL-1β inducedpancreatic β-cell death by the L-TAT-IB peptides.

FIG. 8 is a histogram depicting short term inhibition of IL-1β inducedpancreatic β-cell death by the D-TAT-IB peptides.

FIG. 9 is a histogram depicting long term inhibition of IL-1β inducedpancreatic β-cell death by L-TAT-IB1 and D-TAT-IB1 peptides.

FIG. 10 is a histogram depicting inhibition of irradiation induced humancolon cancer WiDr cell death by L-TAT-IB1 and D-TAT-IB1 peptides.

FIG. 11 is an illustration of the modulation of JNK kinase activity byL-TAT, TAT-IB1 and D-TAT-IB1 peptides.

FIG. 12 are graphs depicting the protective effects of the TAT-IB1peptides in mice. Panel A shows the effect of irradiation on weight.Panel B shows the effect of irradiation on oedemus and erythemus status.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based in part on the discovery of cellpermeable peptides that inhibit the activated c-Jun amino terminalkinase (JNK) signaling pathway. These peptides are referred to herein asJNK inhibitor peptides. Additionally, the discovery provides methods andpharmaceutical compositions for treating pathophysiologies associatedwith JNK signaling.

JNK inhibitor peptides were identified by inspecting sequence alignmentsbetween kJNK Binding Domains in various insulin binding (IB) proteins.The results of this alignment are shown in FIGS. 1A-1C. FIG. 1A depictsthe region of highest homology between the JBDs of IB1, IB2, c-Jun andATF2. Panel B depicts the amino acid sequence alignment of the JBDs ofIB1 and IB2. Fully conserved residues are indicated by asterisks, whileresidues changed to Ala in the GFP-JBD_(23Mut) vector are indicated byopen circles. FIG. 1C shows the amino acid sequences of chimericproteins that include a JNK inhibitor peptide domain and a traffickingdomain. In the example shown, the trafficking domain is derived from thehuman immunodeficiency virus (HIV) TAT polypeptide, and the JNKinhibitor peptide is derived from an IB1 polypeptide. Human, mouse, andrat sequences are identical in Panels B and C.

Sequence comparison between the JNK binding domains of IB1 [SEQ ID NO:17], IB2 [SEQ ID NO: 18], c-Jun [SEQ ID NO: 19] and ATF2 [SEQ ID NO: 20]revealed a partially conserved 8 amino acid sequence (FIG. 1A). Acomparison of the JBDs of IB1 and IB2 further revealed two blocks ofseven and three amino acids that are highly conserved between the twosequences. These two blocks are contained within a peptide sequence of23 amino acids in IB1 [SEQ ID NO: 1] and 21 amino acids in IB2 [SEQ IDNO: 2].

The JNK inhibitor peptides of the invention can be used in any situationin which inhibition of JNK activity is desired. This can include invitro applications, ex vivo, and in vivo applications. As JNKs and allits isoforms participate in the development and establishment ofpathological states or in pathways, the JNK peptides can be used toprevent or inhibit the occurrence of such pathological states. Thisincludes prevention and treatment of diseases and prevention andtreatment of conditions secondary to therapeutic actions. For example,the peptides of the present invention can be used to treat or prevent,e.g., diabetes, ionizing radiation, immune responses (includingautoimmune diseases), ischemia/reperfusion injuries, heart andcardiovascular hypertrophies, and some cancers (e.g., Bcr-Abltransformation).

The peptides can also be used to inhibit expression of genes whoseexpression increases in the presence of an active JNK polypeptide. Thesegenes and gene products includes, e.g., proinflammatory cytokines. Suchcytokines are found in all forms of inflammatory, auto-inflammatory,immune and autoimmune diseases, degenerative diseases, myopathies,cardiomyopathies, and graft rejection.

The JNK inhibitor peptides described herein can also be used to treat orprevent effects associated with cellular shear stress, such as inpathological states induced by arterial hypertension, including cardiachypertrophy and arteriosclerotic lesions, and at bifurcations of bloodvessels, and the like; ionizing radiation, as used in radiotherapy andUV lights; free radicals; DNA damaging agents, includingchemotherapeutic drugs; oncogenic transformation; ischemia andreperfusion; hypoxia; and hypo- and hyperthermia.

The polynucleotides provided by the present invention can be used toexpress recombinant peptides for analysis, characterization ortherapeutic use; as markers for tissues in which the correspondingpeptides is preferentially expressed (either constitutively or at aparticular stage of tissue differentiation or development or in diseasestates). Other uses for the nucleic acids include, e.g., molecularweight markers in gel electrophoresis-based analysiss of nucleic acids.

The JNK inhibitor peptides disclosed herein are presented in Table 1.The table presents the name of the JNK inhibitor peptide, as well as itssequence identifier number, length, and amino acid sequence.

TABLE 1 SEQ PEPTIDE NAME ID AA Sequence L-IB1 1 23DTYRPKRPTT LNLFPQVPRS QDT L-IB2 2 21 EEPHKHRPTT LRLTTLGAQD S D-IB1 3 23TDQSRPVQPF LNLTTPRKPR YTD D-IB2 4 21 SDQAGLTTLR LTTPRHKHPE EL-IB (generic) 5 19 XRPTTLXLXX XXXXXQDS/TX D-IB (generic) 6 19XS/TDQXXXXXX XLXLTTPRX L-TAT 7 10 GRKKRRQRRR D-TAT 8 10 RRRQRRKKRGL-generic-TAT 9 17 XXXXRKKRRQ RRRXXXX D-generic-TAT 10 17XXXXRRRQRR KKRXXXX L-TAT-IB1 11 35GRKKRRQRRR PPDTYRPKRP TTLNLFPQVP RSQDT L-TAT-IB2 12 33GRKKRRQRRR PPEEPHKHRP TTLRLTTLGA QDS L-TAT-IB (generic) 13 42XXXXXXXRKK RRQRRRXXXX XXXXRPTTLX LXXXXXXXQD S/TX D-TAT-IB1 14 35TDQSRPVQPF LNLTTPRKPR YTDPPRRRQR RKKRG D-TAT-IB2 15 33SDQAGLTTLR LTTPRHKHPE EPPRRRQRRK KRG D-TAT-IB (generic) 16 42XT/SDQXXXXXX XLXLTTPRXX XXXXXXRRRQ RRKKRXXXXX XX IB1-long 17 29PGTGCGDTYR PKRPTTLNLF PQVPRSQDT IB2-long 18 27IPSPSVEEPH KHRPTTLRLT TLGAQDS c-Jun 19 29GAYGYSNPKI LKQSMTLNLA DPVGNLKPH ATF2 20 29TNEDHLAVHK HKHEMTLKFG PARNDSVIV

JNK Inhibitor Peptides

In one aspect, the invention provides a JNK inhibitor peptide. Noparticular length is implied by the term “peptide.” In some embodiments,the JNK-inhibitor peptide is less than 280 amino acids in length, e.g.,less than or equal to 150, 100, 75, 50, 35, or 25 amino acids in length.In various embodiment, the JNK-binding inhibitor peptide includes theamino acid sequence of one or more of SEQ ID NOs: 1-6. In oneembodiment, the JNK inhibitor peptide peptides bind JNK. In anotherembodiment the peptide inhibits the activation of at least one JNKactivated transcription factor, e.g. c-Jun, ATF2 or Elk1.

Examples of JNK inhibitor peptides include a peptide which includes (inwhole or in part) the sequence NH ₂-DTYRPKRPTTLNLFPQVPRSQDT-COOH [SEQ IDNO:1]. In another embodiment, the peptide includes the sequence NH₂-EEPHKHRPTTLRLTTLGAQDS-COOH [SEQ ID NO:2]

The JNK inhibitor peptides can be polymers of L-amino acids, D-aminoacids, or a combination of both. For example, in various embodiments,the peptides are D retro-inverso peptides. The term “retro-inversoisomer” refers to an isomer of a linear peptide in which the directionof the sequence is reversed and the chirality of each amino acid residueis inverted. See, e.g., Jameson et al., Nature, 368, 744-746 (1994);Brady et al., Nature, 368, 692-693 (1994). The net result of combiningD-enantiomers and reverse synthesis is that the positions of carbonyland amino groups in each amide bond are exchanged, while the position ofthe side-chain groups at each alpha carbon is preserved. Unlessspecifically stated otherwise, it is presumed that any given L-aminoacid sequence of the invention may be made into an D retro-inversopeptide by synthesizing a reverse of the sequence for the correspondingnative L-amino acid sequence.

In one embodiment, a D retro-inverso peptide has the sequence NH₂-TDQSRPVQPFLNLTTPRKPRYTD-COOH [SEQ ID NO:3]. In another embodiment theD retro-inverso peptide has the sequence NH ₂-SDQAGLTTLRLTTPRHKHPEE-COOH[SEQ ID NO: 4]. It has been unexpectedly found that D-retro-inversoTAT-IB peptides have a variety of useful properties. For example, D-TATand D-TAT-IB peptides enter cells as efficiently as L-TAT and L-TAT-IBpeptides, and D-TAT and D-TAT-IB peptides are more stable than thecoresponding L-peptides. Further, while D-TAT-IB1 are ˜10-20 fold lessefficient in inhibiting JNK than L-TAT-IB, they are ˜50 fold more stablein vivo. Finally, as is discussed further below, D-TAT-IB peptidesprotect interleukin-1 treated and ionizing irradiated cells fromapoptosis.

In another embodiment, a JNK inhibitor peptide according to theinvention includes the amino acid sequence NH₂-X_(n)-RPTTLXLXXXXXXXQDS/T-X_(n)-COOH [SEQ ID NO: 5, and residues 17-42of L-TAT-IB, SEQ ID NO: 13, as shown in FIG. 2]. As used herein, X_(n)may be zero residues in length, or may be a contiguous stretch ofpeptide residues derived from SEQ ID NOS:1-2, preferably a stretch ofbetween 1 and 7 amino acids in length, or may be 10, 20, 30 or moreamino acids in length. The single residue represented by S/T may beeither Ser or Thr in the generic sequence. In a further embodiment, thegeneric B3 peptide may be a D retro-inverso peptide having the sequenceNH ₂-X_(n)-S/TDQXXXXXXXLTTPR-X_(n)-COOH [SEQ ID NO: 6, and residues17-42 of L-TAT-IB, SEQ ID NO:16, as shown in FIG. 2].

JNK-inhibitor peptides may be obtained or produced by methods well-knownin the art, e.g. chemical synthesis, genetic engineering methods asdiscussed below. For example, a peptide corresponding to a portion of aJNK inhibitor peptide including a desired region or domain, or thatmediates the desired activity in vitro, may be synthesized by use of apeptide synthesizer.

A candidate JNK inhibitor peptide may also be analyzed by hydrophilicityanalysis (see, e.g., Hopp and Woods, 1981. Proc Natl Acad Sci USA 78:3824-3828) that can be utilized to identify the hydrophobic andhydrophilic regions of the peptides, thus aiding in the design ofsubstrates for experimental manipulation, such as in bindingexperiments, antibody synthesis. Secondary structural analysis may alsobe performed to identify regions of a JNK inhibitor peptide that assumespecific structural motifs. See e.g., Chou and Fasman, 1974. Biochem 13:222-223. Manipulation, translation, secondary structure prediction,hydrophilicity and hydrophobicity profiles, open reading frameprediction and plotting, and determination of sequence homologies can beaccomplished using computer software programs available in the art.Other methods of structural analysis including, e.g., X-raycrystallography (see, e.g., Engstrom, 1974. Biochem Exp Biol 11: 7-13);mass spectroscopy and gas chromatography (see, e.g., METHODS IN PROTEINSCIENCE, 1997. J. Wiley and Sons, New York, N.Y.) and computer modeling(see, e.g., Fletterick and Zoller, eds., 1986. Computer Graphics andMolecular Modeling, In: CURRENT COMMUNICATIONS IN MOLECULAR BIOLOGY,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) may alsobe employed.

The present invention additionally relates to nucleic acids that encodeJNK-binding peptides having L-form amino acids, e.g., those L-peptidesindicated in Table 1, as well as the complements of these sequences.Suitable sources of nucleic acids encoding JNK inhibitor peptidesinclude the human IB1 nucleic acid (and the encoded protein sequences)available as GenBank Accession Nos. AF074091 and AAD20443, respectively.Other sources include rat IB1 nucleic acid and protein sequences areshown in GenBank Accession No. AF108959 and AAD22543, respectively, andare incorporated herein by reference in their entirety. Human IB2nucleic acid and protein sequences are shown in GenBank Accession NoAF218778 and is also incorporated herein by reference in their entirety.

Nucleic acids encoding the JNK inhibitor peptides may be obtained by anymethod known in the art (e.g., by PCR amplification using syntheticprimers hybridizable to the 3′- and 5′-termini of the sequence and/or bycloning from a cDNA or genomic library using an oligonucleotide sequencespecific for the given gene sequence).

For recombinant expression of one or more JNK inhibitor peptides, thenucleic acid containing all or a portion of the nucleotide sequenceencoding the peptide may be inserted into an appropriate expressionvector (i.e., a vector that contains the necessary elements for thetranscription and translation of the inserted peptide coding sequence).In some embodiments, the regulatory elements are heterologous (i.e., notthe native gene promoter). Alternately, the necessary transcriptionaland translational signals may also be supplied by the native promoterfor the genes and/or their flanking regions.

A variety of host-vector systems may be utilized to express the peptidecoding sequence(s). These include, but are not limited to: (i) mammaliancell systems that are infected with vaccinia virus, adenovirus, and thelike; (ii) insect cell systems infected with baculovirus and the like;(iii) yeast containing yeast vectors or (iv) bacteria transformed withbacteriophage, DNA, plasmid DNA, or cosmid DNA. Depending upon thehost-vector system utilized, any one of a number of suitabletranscription and translation elements may be used.

Promoter/enhancer sequences within expression vectors may utilize plant,animal, insect, or fungus regulatory sequences, as provided in theinvention. For example, promoter/enhancer elements can b used from yeastand other fungi (e.g., the GAL4 promoter, the alcohol dehydrogenasepromoter, the phosphoglycerol kinase promoter, the alkaline phosphatasepromoter). Alternatively, or in addition, they may include animaltranscriptional control regions, e.g., (i) the insulin gene controlregion active within pancreatic β-cells (see, e.g., Hanahan, et al.,1985. Nature 315: 115-122); (ii) the immunoglobulin gene control regionactive within lymphoid cells (see, e.g., Grosschedl, et al., 1984. Cell38: 647-658); (iii) the albumin gene control region active within liver(see, e.g., Pinckert, et al., 1987. Genes and Dev 1: 268-276; (iv) themyelin basic protein gene control region active within brainoligodendrocyte cells (see, e.g., Readhead, et al., 1987. Cell 48:703-712); and (v) the gonadotropin-releasing hormone gene control regionactive within the hypothalamus (see, e.g., Mason, et al., 1986. Science234: 1372-1378), and the like.

Expression vectors or their derivatives include, e.g. human or animalviruses (e.g., vaccinia virus or adenovirus); insect viruses (e.g.,baculovirus); yeast vectors; bacteriophage vectors (e.g., lambda phage);plasmid vectors and cosmid vectors.

A host cell strain may be selected that modulates the expression ofinserted sequences of interest, or modifies or processes expressedpeptides encoded by the sequences in the specific manner desired. Inaddition, expression from certain promoters may be enhanced in thepresence of certain inducers in a selected host strain; thusfacilitating control of the expression of a genetically-engineeredpeptides. Moreover, different host cells possess characteristic andspecific mechanisms for the translational and post-translationalprocessing and modification (e.g., glycosylation, phosphorylation, andthe like) of expressed peptides. Appropriate cell lines or host systemsmay thus be chosen to ensure the desired modification and processing ofthe foreign peptide is achieved. For example, peptide expression withina bacterial system can be used to produce an unglycosylated corepeptide; whereas expression within mammalian cells ensures “native”glycosylation of a heterologous peptide.

Also included in the invention are derivatives, fragments, homologs,analogs and variants of JNK inhibitor peptides and nucleic acidsencoding these peptides. For nucleic acids, derivatives, fragments, andanalogs provided herein are defined as sequences of at least 6(contiguous) nucleic acids, and which have a length sufficient to allowfor specific hybridization. For amino acids, derivatives, fragments, andanalogs provided herein are defined as sequences of at least 4(contiguous) amino acids, a length sufficient to allow for specificrecognition of an epitope.

The length of the fragments are less than the length of thecorresponding full-length nucleic acid or polypeptide from which the JNKinhibitor peptide, or nucleic acid encoding same, is derived.Derivatives and analogs may be full length or other than full length, ifthe derivative or analog contains a modified nucleic acid or amino acid.Derivatives or analogs of the JNK inhibitor peptides include, e.g.,molecules including regions that are substantially homologous to thepeptides, in various embodiments, by at least about 30%, 50%, 70%, 80%,or 95%, 98%, or even 99%, identity over an amino acid sequence ofidentical size or when compared to an aligned sequence in which thealignment is done by a computer homology program known in the art. Forexample sequence identity can be measured using sequence analysissoftware (Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705), with the default parameters therein.

In the case of polypeptide sequences, which are less than 100% identicalto a reference sequence, the non-identical positions are preferably, butnot necessarily, conservative substitutions for the reference sequence.Conservative substitutions typically include substitutions within thefollowing groups: glycine and alanine; valine, isoleucine, and leucine;aspartic acid and glutamic acid; asparagine and glutamine; serine andthreonine; lysine and arginine; and phenylalanine and tyrosine. Thus,included in the invention are peptides having mutated sequences suchthat they remain homologous, e.g. in sequence, in function, and inantigenic character or other function, with a protein having thecorresponding parent sequence. Such mutations can, for example, bemutations involving conservative amino acid changes, e.g., changesbetween amino acids of broadly similar molecular properties. Forexample, interchanges within the aliphatic group alanine, valine,leucine and isoleucine can be considered as conservative. Sometimessubstitution of glycine for one of these can also be consideredconservative. Other conservative interchanges include those within thealiphatic group aspartate and glutamate; within the amide groupasparagine and glutamine; within the hydroxyl group serine andthreonine; within the aromatic group phenylalanine, tyrosine andtryptophan; within the basic group lysine, arginine and histidine; andwithin the sulfur-containing group methionine and cysteine. Sometimessubstitution within the group methionine and leucine can also beconsidered conservative. Preferred conservative substitution groups areaspartate-glutamate; asparagine-glutamine; valine-leucine-isoleucine;alanine-valine; phenylalanine-tyrosine; and lysine-arginine.

Where a particular polypeptide is said to have a specific percentidentity to a reference polypeptide of a defined length, the percentidentity is relative to the reference peptide. Thus, a peptide that is50% identical to a reference polypeptide that is 100 amino acids longcan be a 50 amino acid polypeptide that is completely identical to a 50amino acid long portion of the reference polypeptide. It might also be a100 amino acid long polypeptide, which is 50% identical to the referencepolypeptide over its entire length. Of course, other polypeptides willmeet the same criteria.

The invention also encompasses allelic variants of the disclosedpolynucleotides or peptides; that is, naturally-occurring alternativeforms of the isolated polynucleotide that also encode peptides that areidentical, homologous or related to that encoded by the polynucleotides.Alternatively, non-naturally occurring variants may be produced bymutagenesis techniques or by direct synthesis.

Species homologs of the disclosed polynucleotides and peptides are alsoprovided by the present invention. “Variant” refers to a polynucleotideor polypeptide differing from the polynucleotide or polypeptide of thepresent invention, but retaining essential properties thereof.Generally, variants are overall closely similar, and in many regions,identical to the polynucleotide or polypeptide of the present invention.The variants may contain alterations in the coding regions, non-codingregions, or both.

In some embodiments, altered sequences include insertions such that theoverall amino acid sequence is lengthened while the protein retainstrafficking properties. Additionally, altered sequences may includerandom or designed internal deletions that shorten the overall aminoacid sequence while the protein retains transport properties.

The altered sequences can additionally or alternatively be encoded bypolynucleotides that hybridize under stringent conditions with theappropriate strand of the naturally-occurring polynucleotide encoding apolypeptide or peptide from which the JNK inhibitor peptide is derived.The variant peptide can be tested for JNK-binding and modulation ofJNK-mediated activity using the herein described assays. ‘Stringentconditions’ are sequence dependent and will be different in differentcircumstances. Generally, stringent conditions can be selected to beabout 5° C. lower than the thermal melting point (T_(M)) for thespecific sequence at a defined ionic strength and pH. The T_(M) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Typically,stringent conditions will be those in which the salt concentration is atleast about 0.02 molar at pH 7 and the temperature is at least about 60°C. As other factors may affect the stringency of hybridization(including, among others, base composition and size of the complementarystrands), the presence of organic solvents and the extent of basemismatching, the combination of parameters is more important than theabsolute measure of any one.

High stringency can include, e.g., Step 1: Filters containing DNA arepretreated for 8 hours to overnight at 65° C. in buffer composed of6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll,0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Step 2: Filters arehybridized for 48 hours at 65° C. in the above prehybridization mixtureto which is added 100 mg/ml denatured salmon sperm DNA and 5-20×10⁶ cpmof ³²P-labeled probe. Step 3: Filters are washed for 1 hour at 37° C. ina solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA.This is followed by a wash in 0.1×SSC at 50° C. for 45 minutes. Step 4:Filters are autoradiographed. Other conditions of high stringency thatmay be used are well known in the art. See, e.g., Ausubel et al.,(eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley andSons, NY; and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORYMANUAL, Stockton Press, NY.

Moderate stringency conditions can include the following: Step 1:Filters containing DNA are pretreated for 6 hours at 55° C. in asolution containing 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 mg/mldenatured salmon sperm DNA. Step 2: Filters are hybridized for 18-20hours at 55° C. in the same solution with 5-20×10⁶ cpm ³²P-labeled probeadded. Step 3: Filters are washed at 37° C. for 1 hour in a solutioncontaining 2×SSC, 0.1% SDS, then washed twice for 30 minutes at 60° C.in a solution containing 1×SSC and 0.1% SDS. Step 4: Filters are blotteddry and exposed for autoradiography. Other conditions of moderatestringency that may be used are well-known in the art. See, e.g.,Ausubel et al., (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley and Sons, NY; and Kriegler, 1990, GENE TRANSFER ANDEXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.

Low stringency can include: Step 1: Filters containing DNA arepretreated for 6 hours at 40° C. in a solution containing 35% formamide,5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1%BSA, and 500 μg/ml denatured salmon sperm DNA. Step 2: Filters arehybridized for 18-20 hours at 40° C. in the same solution with theaddition of 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml salmon spermDNA, 10% (wt/vol) dextran sulfate, and 5-20×10⁶ cpm ³²P-labeled probe.Step 3: Filters are washed for 1.5 hours at 55° C. in a solutioncontaining 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. Thewash solution is replaced with fresh solution and incubated anadditional 1.5 hours at 60° C. Step 4: Filters are blotted dry andexposed for autoradiography. If necessary, filters are washed for athird time at 65-68° C. and reexposed to film. Other conditions of lowstringency that may be used are well known in the art (e.g., as employedfor cross-species hybridizations). See, e.g., Ausubel et al., (eds.),1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons, NY;and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL,Stockton Press, NY.

Chimeric Peptides Including a JNK Inhibitor Domain and a TraffickingDomain

In another aspect the invention provides a chimeric peptide thatincludes a first and second domain. The first domain includes atrafficking sequence, while the second domain includes a JNK inhibitorsequence linked by a covalent bond, e.g. peptide bond, to the firstdomain. The first and second domains can occur in any order in thepeptide, and the peptide can include one or more of each domain.

A trafficking sequence is any sequence of amino acids that directs apeptide in which it is present to a desired cellular destination. Thus,the trafficking sequence can direct the peptide across the plasmamembrane, e.g., from outside the cell, through the plasma membrane, andinto the cytoplasm. Alternatively, or in addition, the traffickingsequence can direct the peptide to a desired location within the cell,e.g., the nucleus, the ribosome, the ER, a lysosome, or peroxisome.

In some embodiments, the trafficking peptide is derived from a knownmembrane-translocating sequence. For example, the trafficking peptidemay include sequences from the human immunodeficiency virus (HIV) 1 TATprotein. This protein is described in, e.g., U.S. Pat. Nos. 5,804,604and 5,674,980, each incorporated herein by reference. The JNK inhibitorpeptide may be linked to some or all of the entire 86 amino acids thatmake up the TAT protein. For example, a functionally effective fragmentor portion of a TAT protein that has fewer than 86 amino acids, whichexhibits uptake into cells, and optionally uptake into the cell nucleus,can be used. In one embodiment, the fragment includes a peptidecontaining TAT residues 48-57, e.g. NH ₂-GRKKRRQRRR-COOH [SEQ ID NO: 7]or a generic TAT sequence NH ₂-X_(n)-RKKRRQRRR-X_(n)-COOH [SEQ ID NO:9]. A TAT peptide that includes the region that mediates entry anduptake into cells can be further defined using known techniques. See,e.g., Franked et al., Proc. Natl. Acad. Sci, USA 86: 7397-7401 (1989).

The TAT sequence may be linked either to the N-terminal or theC-terminal end of JNK inhibitor sequence. A hinge of two prolineresidues may be added between the TAT and JNK inhibitor peptide tocreate the full fusion peptide. For example, L-amino acid fusionpeptides may be the L-TAT-IB1 peptide [SEQ ID NO:11], the L-TAT-IB2peptide [SEQ ID NO:12], or the generic L-TAT-IB peptide [SEQ ID NO:13].D retro-inverso fusion peptides may be the D-TAT-IB1 peptide [SEQ IDNO:14], the D-TAT-IB2 peptide [SEQ ID NO:15], or the generic D-TAT-IBpeptide [SEQ ID NO:16]. The TAT peptide may be a D retro-inverso peptidehaving the sequence NH ₂-X_(n)-RRRQRRKKR-X_(n)-COOH [SEQ ID NO:10]. InSEQ ID NOs:5-6,9-10, 13 and 16, the number of “X” residues is notlimited to the one depicted, and may vary as described above.

The trafficking sequence can be a single (i.e., continuous) amino acidsequence present in the TAT sequence. Alternatively it can be two ormore amino acid sequences, which are present in TAT protein, but in thenaturally-occurring protein are separated by other amino acid sequences.As used herein, TAT protein includes a naturally-occurring amino acidsequence that is the same as that of naturally-occurring TAT protein, orits functional equivalent protein or functionally equivalent fragmentsthereof (peptides). Such functional equivalent proteins or functionallyequivalent fragments possess uptake activity into the cell and into thecell nucleus that is substantially similar to that ofnaturally-occurring TAT protein. TAT protein can be obtained fromnaturally-occurring sources or can be produced using genetic engineeringtechniques or chemical synthesis.

The amino acid sequence of naturally-occurring HIV TAT protein can bemodified, for example, by addition, deletion and/or substitution of atleast one amino acid present in the naturally-occurring TAT protein, toproduce modified TAT protein (also referred to herein as TAT protein).Modified TAT protein or TAT peptide analogs with increased or decreasedstability can be produced using known techniques. In some embodimentsTAT proteins or peptides include amino acid sequences that aresubstantially similar, although not identical, to that ofnaturally-occurring TAT protein or portions thereof. In addition,cholesterol or other lipid derivatives can be added to TAT protein toproduce a modified TAT having increased membrane solubility.

Variants of the TAT protein can be designed to modulate intracellularlocalization of TAT-JNK inhibitor peptide. When added exogenously, suchvariants are designed such that the ability of TAT to enter cells isretained (i.e., the uptake of the variant TAT protein or peptide intothe cell is substantially similar to that of naturally-occurring HIVTAT). For example, alteration of the basic region thought to beimportant for nuclear localization (see, e.g., Dang and Lee, J. Biol.Chem. 264: 18019-18023 (1989); Hauber et al., J. Virol. 63: 1181-1187(1989); Ruben et al., J. Virol. 63: 1-8 (1989)) can result in acytoplasmic location or partially cytoplasmic location of TAT, andtherefore, of the JNK inhibitor peptide. Alternatively, a sequence forbinding a cytoplasmic or any other component or compartment (e.g.,endoplasmic reticule, mitochondria, gloom apparatus, lysosomalvesicles,) can be introduced into TAT in order to retain TAT and the JNKinhibitor peptide in the cytoplasm or any other compartment to conferregulation upon uptake of TAT and the JNK inhibitor peptide.

Other sources for the trafficking peptide include, e.g., VP22 (describedin, e.g., WO 97/05265; Elliott and O'Hare, Cell 88: 223-233 (1997)), ornon-viral proteins (Jackson et al, Proc. Natl. Acad. Sci. USA 89:10691-10695 (1992)).

The JNK inhibitor sequence and the trafficking sequence can be linked bychemical coupling in any suitable manner known in the art. Many knownchemical cross-linking methods are non-specific, i.e.; they do notdirect the point of coupling to any particular site on the transportpolypeptide or cargo macromolecule. As a result, use of non-specificcross-linking agents may attack functional sites or sterically blockactive sites, rendering the conjugated proteins biologically inactive.

One way to increasing coupling specificity is to directly chemicalcoupling to a functional group found only once or a few times in one orboth of the polypeptides to be cross-linked. For example, in manyproteins, cysteine, which is the only protein amino acid containing athiol group, occurs only a few times. Also, for example, if apolypeptide contains no lysine residues, a cross-linking reagentspecific for primary amines will be selective for the amino terminus ofthat polypeptide. Successful utilization of this approach to increasecoupling specificity requires that the polypeptide have the suitablyrare and reactive residues in areas of the molecule that may be alteredwithout loss of the molecule's biological activity.

Cysteine residues may be replaced when they occur in parts of apolypeptide sequence where their participation in a cross-linkingreaction would otherwise likely interfere with biological activity. Whena cysteine residue is replaced, it is typically desirable to minimizeresulting changes in polypeptide folding. Changes in polypeptide foldingare minimized when the replacement is chemically and sterically similarto cysteine. For these reasons, serine is preferred as a replacement forcysteine. As demonstrated in the examples below, a cysteine residue maybe introduced into a polypeptide's amino acid sequence for cross-linkingpurposes. When a cysteine residue is introduced, introduction at or nearthe amino or carboxy terminus is preferred. Conventional methods areavailable for such amino acid sequence modifications, whether thepolypeptide of interest is produced by chemical synthesis or expressionof recombinant DNA.

Coupling of the two constituents can be accomplished via a coupling orconjugating agent. There are several intermolecular cross-linkingreagents which can be utilized, See for example, Means and Feeney,CHEMICAL MODIFICATION OF PROTEINS, Holden-Day, 1974, pp. 39-43. Amongthese reagents are, for example, J-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) or N,N′-(1,3-phenylene) bismaleimide (both of whichare highly specific for sulfhydryl groups and form irreversiblelinkages); N,N′-ethylene-bis-(iodoacetamide) or other such reagenthaving 6 to 11 carbon methylene bridges (which relatively specific forsulfhydryl groups); and 1,5-difluoro-2,4-dinitrobenzene (which formsirreversible linkages with amino and tyrosine groups). Othercross-linking reagents useful for this purpose include:p,p′-difluoro-m,m′-dinitrodiphenylsulfone (which forms irreversiblecross-linkages with amino and phenolic groups); dimethyl adipimidate(which is specific for amino groups); phenol-1,4-disulfonylchloride(which reacts principally with amino groups); hexamethylenediisocyanateor diisothiocyanate, or azophenyl-p-diisocyanate (which reactsprincipally with amino groups); glutaraldehyde (which reacts withseveral different side chains) and disdiazobenzidine (which reactsprimarily with tyrosine and histidine).

Cross-linking reagents may be homobifunctional, i.e., having twofunctional groups that undergo the same reaction. A preferredhomobifunctional cross-linking reagent is bismaleimidohexane (“BMH”).BMH contains two maleimide functional groups, which react specificallywith sulfhydryl-containing compounds under mild conditions (pH 6.5-7.7).The two maleimide groups are connected by a hydrocarbon chain.Therefore, BMH is useful for irreversible cross-linking of polypeptidesthat contain cysteine residues.

Cross-linking reagents may also be heterobifunctional.Heterobifunctional cross-linking agents have two different functionalgroups, for example an amine-reactive group and a thiol-reactive group,that will cross-link two proteins having free amines and thiols,respectively. Examples of heterobifunctional cross-linking agents aresuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (“SMCC”),m-maleimidobenzoyl-N-hydroxysuccinimide ester (“MBS”), and succinimide4-(p-maleimidophenyl) butyrate (“SMPB”), an extended chain analog ofMBS. The succinimidyl group of these cross-linkers reacts with a primaryamine, and the thiol-reactive maleimide forms a covalent bond with thethiol of a cysteine residue.

Cross-linking reagents often have low solubility in water. A hydrophilicmoiety, such as a sulfonate group, may be added to the cross-linkingreagent to improve its water solubility. Sulfo-MBS and sulfo-SMCC areexamples of cross-linking reagents modified for water solubility.

Many cross-linking reagents yield a conjugate that is essentiallynon-cleavable under cellular conditions. However, some cross-linkingreagents contain a covalent bond, such as a disulfide, that is cleavableunder cellular conditions. For example, Traut's reagent, dithiobis(succinimidylpropionate) (“DSP”), and N-succinimidyl 3-(2-pyridyldithio)propionate (“SPDP”) are well-known cleavable cross-linkers. The use of acleavable cross-linking reagent permits the cargo moiety to separatefrom the transport polypeptide after delivery into the target cell.Direct disulfide linkage may also be useful.

Numerous cross-linking reagents, including the ones discussed above, arecommercially available. Detailed instructions for their use are readilyavailable from the commercial suppliers. A general reference on proteincross-linking and conjugate preparation is: Wong, CHEMISTRY OF PROTEINCONJUGATION AND CROSS-LJNKING, CRC Press (1991).

Chemical cross-linking may include the use of spacer arms. Spacer armsprovide intramolecular flexibility or adjust intramolecular distancesbetween conjugated moieties and thereby may help preserve biologicalactivity. A spacer arm may be in the form of a polypeptide moiety thatincludes spacer amino acids, e.g. proline. Alternatively, a spacer armmay be part of the cross-linking reagent, such as in “long-chain SPDP”(Pierce Chem. Co., Rockford, Ill., cat. No. 21651 H).

Alternatively, the chimeric peptide can be produced as a fusion peptidethat includes the trafficking sequence and the JNK inhibitor sequencewhich can conveniently be expressed in known suitable host cells. Fusionpeptides, as described herein, can be formed and used in ways analogousto or readily adaptable from standard recombinant DNA techniques, asdescribe above.

Production of Antibodies Specific for JNK Inhibitor Peptides

JNK inhibitor peptides, including chimeric peptides including the JNKinhibitor peptides (e.g., peptides including the amino acid sequencesshown in Table 1), as well peptides, or derivatives, fragments, analogsor homologs thereof, may be utilized as immunogens to generateantibodies that immunospecifically-bind these peptide components. Suchantibodies include, e.g., polyclonal, monoclonal, chimeric, singlechain, Fab fragments and a Fab expression library. In a specificembodiment, antibodies to human peptides are disclosed. In anotherspecific embodiment, fragments of the JNK inhibitor peptides are used asimmunogens for antibody production. Various procedures known within theart may be used for the production of polyclonal or monoclonalantibodies to a JNK inhibitor peptide, or derivative, fragment, analogor homolog thereof.

For the production of polyclonal antibodies, various host animals may beimmunized by injection with the native peptide, or a synthetic variantthereof, or a derivative of the foregoing. Various adjuvants may be usedto increase the immunological response and include, but are not limitedto, Freund's (complete and incomplete), mineral gels (e.g., aluminumhydroxide), surface active substances (e.g., lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.) andhuman adjuvants such as Bacille Calmette-Guerin and Corynebacteriumparvum.

For preparation of monoclonal antibodies directed towards a JNKinhibitor peptide, or derivatives, fragments, analogs or homologsthereof, any technique that provides for the production of antibodymolecules by continuous cell line culture may be utilized. Suchtechniques include, but are not limited to, the hybridoma technique(see, Kohler and Milstein, 1975. Nature 256: 495-497); the triomatechnique; the human B-cell hybridoma technique (see, Kozbor, et al.,1983. Immunol Today 4: 72) and the EBV hybridoma technique to producehuman monoclonal antibodies (see, Cole, et al., 1985. In: MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Humanmonoclonal antibodies may be utilized in the practice of the presentinvention and may be produced by the use of human hybridomas (see, Cote,et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforminghuman B-cells with Epstein Barr Virus in vitro (see, Cole, et al., 1985.In: Monoclonal Antibodies and Cancer Therapy (Alan R. Liss, Inc., pp.77-96).

According to the invention, techniques can be adapted for the productionof single-chain antibodies specific to a JNK inhibitor peptide (see,e.g., U.S. Pat. No. 4,946,778). In addition, methodologies can beadapted for the construction of Fab expression libraries (see, e.g.,Huse, et al., 1989. Science 246: 1275-1281) to allow rapid and effectiveidentification of monoclonal Fab fragments with the desired specificityfor a JNK inhibitor peptide or derivatives, fragments, analogs orhomologs thereof. Non-human antibodies can be “humanized” by techniqueswell known in the art. See e.g., U.S. Pat. No. 5,225,539. Antibodyfragments that contain the idiotypes to a JNK inhibitor peptide may beproduced by techniques known in the art including, e.g., (i) an F(ab′)₂fragment produced by pepsin digestion of an antibody molecule; (ii) anFab fragment generated by reducing the disulfide bridges of an F(ab′)₂fragment; (iii) an Fab fragment generated by the treatment of theantibody molecule with papain and a reducing agent and (iv) Fvfragments.

In one embodiment, methodologies for the screening of antibodies thatpossess the desired specificity include, but are not limited to,enzyme-linked immunosorbent assay (ELISA) and otherimmunologically-mediated techniques known within the art. In a specificembodiment, selection of antibodies that are specific to a particulardomain of a JNK inhibitor peptide is facilitated by generation ofhybridomas that bind to the fragment of a JNK inhibitor peptidepossessing such a domain. Antibodies that are specific for a domainwithin a JNK inhibitor peptide, or derivative, fragments, analogs orhomologs thereof, are also provided herein.

The anti-JNK inhibitor peptide antibodies may be used in methods knownwithin the art relating to the localization and/or quantitation of a JNKinhibitor peptide (e.g., for use in measuring levels of the peptidewithin appropriate physiological samples, for use in diagnostic methods,for use in imaging the peptide, and the like). In a given embodiment,antibodies for the JNK inhibitor peptides, or derivatives, fragments,analogs or homologs thereof that contain the antibody derived bindingdomain, are utilized as pharmacologically active compounds [hereinafter“Therapeutics”].

Methods of Treating or Preventing Disorders Associated Undesired JNKActivity

Also included in the invention also are methods of treatingcell-proliferative disorders associated with JNK activation in a subjectby administering to a subject a biologically-active therapeutic compound(hereinafter “Therapeutic”). The subject can be e.g., any mammal, e.g.,a human, a primate, mouse, rat, dog, cat, cow, horse, pig.

The Therapeutics include, e.g.: (i) any one or more of the JNK inhibitorpeptides, and derivative, fragments, analogs and homologs thereof; (ii)antibodies directed against the JNK inhibitor peptides; (iii) nucleicacids encoding a JNK inhibitor peptide, and derivatives, fragments,analogs and homologs thereof; (iv) antisense nucleic acids to sequencesencoding a JNK inhibitor peptide, and (v) modulators (i.e., inhibitors,agonists and antagonists).

The term “therapeutically effective” means that the amount of inhibitorpeptide, for example, which is used, is of sufficient quantity toameliorate the JNK associated disorder. The term “cell-proliferativedisorder” denotes malignant as well as non-malignant cell populationsthat often appear to differ morphologically and functionally from thesurrounding tissue. For example, the method may be useful in treatingmalignancies of the various organ systems, in which activation of JNKhas often been demonstrated, e.g., lung, breast, lymphoid,gastrointestinal, and genito-urinary tract as well as adenocarcinomaswhich include malignancies such as most colon cancers, renal-cellcarcinoma, prostate cancer, non-small cell carcinoma of the lung, cancerof the small intestine and cancer of the esophagus. Cancers with Bcr-Abloncogenic transformations that clearly require activation of JNK arealso included.

The method is also useful in treating non-malignant orimmunological-related cell-proliferative diseases such as psoriasis,pemphigus vulgaris, Behcet's syndrome, acute respiratory distresssyndrome (ARDS), ischemic heart disease, post-dialysis syndrome,leukemia, rheumatoid arthritis, acquired immune deficiency syndrome,vasculitis, septic shock and other types of acute inflammation, andlipid histiocytosis. Especially preferred are immunopathologicaldisorders. Essentially, any disorder, which is etiologically linked toJNK kinase activity, would be considered susceptible to treatment.

Treatment includes administration of a reagent that modulates JNK kinaseactivity. The term “modulate” includes the suppression of expression ofJNK when it is over-expressed. It also includes suppression ofphosphorylation of c-jun, ATF2 or NFAT4, for example, by using a peptideof any one or more of SEQ ID NOs: 1-6 and SEQ ID NOs: 11-16 as acompetitive inhibitor of the natural c-jun ATF2 and NFAT4 binding sitein a cell. Thus also includes suppression of hetero- and homo-mericcomplexes of transcription factors made up of c-jun, ATF2, or NFAT4 andtheir related partners, such as for example the AP-1 complex that ismade up of c-jun, AFT2 and c-fos. When a cell proliferative disorder isassociated with JNK overexpression, such suppressive JNK inhibitorpeptides can be introduced to a cell. In some instances, “modulate” mayinclude the increase of JNK expression, for example by use of an IBpeptide-specific antibody that blocks the binding of an IB-peptide toJNK, thus preventing JNK inhibition by the IB-related peptide.

The JNK inhibitor, peptides, fusion peptides and nucleic acids of theinvention can be formulated in pharmaceutical compositions. Thesecompositions may comprise, in addition to one of the above substances, apharmaceutically acceptable excipient, carrier, buffer, stabiliser orother materials well known to those skilled in the art. Such materialsshould be non-toxic and should not interfere with the efficacy of theactive ingredient. The precise nature of the carrier or other materialmay depend on the route of administration, e.g. oral, intravenous,cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal orpatch routes.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may include a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally include a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

Whether it is a polypeptide, peptide, or nucleic acid molecule, otherpharmaceutically useful compound according to the present invention thatis to be given to an individual, administration is preferably in a“prophylactically effective amount” or a “therapeutically effectiveamount” (as the case may be, although prophylaxis may be consideredtherapy), this being sufficient to show benefit to the individual. Theactual amount administered, and rate and time-course of administration,will depend on the nature and severity of what is being treated.Prescription of treatment, e.g. decisions on dosage etc, is within theresponsibility of general practitioners and other medical doctors, andtypically takes account of the disorder to be treated, the condition ofthe individual patient, the site of delivery, the method ofadministration and other factors known to practitioners. Examples of thetechniques and protocols mentioned above can be found in REMINGTON'SPHARMACEUTICAL SCIENCES, 16th edition, Osol, A. (ed), 1980.

Alternatively, targeting therapies may be used to deliver the activeagent more specifically to certain types of cell, by the use oftargeting systems such as antibody or cell specific ligands. Targetingmay be desirable for a variety of reasons; for example if the agent isunacceptably toxic, or if it would otherwise require too high a dosage,or if it would not otherwise be able to enter the target cells.

Instead of administering these agents directly, they could be producedin the target cells by expression from an encoding gene introduced intothe cells, e.g. in a viral vector (a variant of the VDEPT technique—seebelow). The vector could be targeted to the specific cells to betreated, or it could contain regulatory elements, which are switched onmore or less selectively by the target cells.

Alternatively, the agent could be administered in a precursor form, forconversion to the active form by an activating agent produced in, ortargeted to, the cells to be treated. This type of approach is sometimesknown as ADEPT or VDEPT; the former involving targeting the activatingagent to the cells by conjugation to a cell-specific antibody, while thelatter involves producing the activating agent, e.g. a JNK inhibitorpeptide, in a vector by expression from encoding DNA in a viral vector(see for example, EP-A-415731 and WO 90/07936).

In a specific embodiment of the present invention, nucleic acids includea sequence that encodes a JNK inhibitor peptide, or functionalderivatives thereof, are administered to modulate activated JNKsignaling pathways by way of gene therapy. In more specific embodiments,a nucleic acid or nucleic acids encoding a JNK inhibitor peptide, orfunctional derivatives thereof, are administered by way of gene therapy.Gene therapy refers to therapy that is performed by the administrationof a specific nucleic acid to a subject. In this embodiment of thepresent invention, the nucleic acid produces its encoded peptide(s),which then serve to exert a therapeutic effect by modulating function ofthe disease or disorder. Any of the methodologies relating to genetherapy available within the art may be used in the practice of thepresent invention. See e.g., Goldspiel, et al., 1993. Clin Pharm 12:488-505.

In a preferred embodiment, the Therapeutic comprises a nucleic acid thatis part of an expression vector expressing any one or more of theIB-related peptides, or fragments, derivatives or analogs thereof,within a suitable host. In a specific embodiment, such a nucleic acidpossesses a promoter that is operably-linked to coding region(s) of aJNK inhibitor peptide. The promoter may be inducible or constitutive,and, optionally, tissue-specific. In another specific embodiment, anucleic acid molecule is used in which coding sequences (and any otherdesired sequences) are flanked by regions that promote homologousrecombination at a desired site within the genome, thus providing forintra-chromosomal expression of nucleic acids. See e.g., Koller andSmithies, 1989. Proc Natl Acad Sci USA 86: 8932-8935.

Delivery of the Therapeutic nucleic acid into a patient may be eitherdirect (i.e., the patient is directly exposed to the nucleic acid ornucleic acid-containing vector) or indirect (i.e., cells are firsttransformed with the nucleic acid in vitro, then transplanted into thepatient). These two approaches are known, respectively, as in vivo or exvivo gene therapy. In a specific embodiment of the present invention, anucleic acid is directly administered in vivo, where it is expressed toproduce the encoded product. This may be accomplished by any of numerousmethods known in the art including, e.g., constructing the nucleic acidas part of an appropriate nucleic acid expression vector andadministering the same in a manner such that it becomes intracellular(e.g., by infection using a defective or attenuated retroviral or otherviral vector; see U.S. Pat. No. 4,980,286); directly injecting nakedDNA; using microparticle bombardment (e.g., a “Gene Gun®; Biolistic,DuPont); coating the nucleic acids with lipids; using associatedcell-surface receptors/transfecting agents; encapsulating in liposomes,microparticles, or microcapsules; administering it in linkage to apeptide that is known to enter the nucleus; or by administering it inlinkage to a ligand predisposed to receptor-mediated endocytosis (see,e.g., Wu and Wu, 1987. J Biol Chem 262: 4429-4432), which can be used to“target” cell types that specifically express the receptors of interest,etc.

An additional approach to gene therapy in the practice of the presentinvention involves transferring a gene into cells in in vitro tissueculture by such methods as electroporation, lipofection, calciumphosphate-mediated transfection, viral infection, or the like.Generally, the method of transfer includes the concomitant transfer of aselectable marker to the cells. The cells are then placed underselection pressure (e.g., antibiotic resistance) so as to facilitate theisolation of those cells that have taken up, and are expressing, thetransferred gene. Those cells are then delivered to a patient. In aspecific embodiment, prior to the in vivo administration of theresulting recombinant cell, the nucleic acid is introduced into a cellby any method known within the art including, e.g., transfection,electroporation, microinjection, infection with a viral or bacteriophagevector containing the nucleic acid sequences of interest, cell fusion,chromosome-mediated gene transfer, microcell-mediated gene transfer,spheroplast fusion, and similar methodologies that ensure that thenecessary developmental and physiological functions of the recipientcells are not disrupted by the transfer. See e.g., Loeffler and Behr,1993. Meth Enzymol 217: 599-618. The chosen technique should provide forthe stable transfer of the nucleic acid to the cell, such that thenucleic acid is expressible by the cell. Preferably, the transferrednucleic acid is heritable and expressible by the cell progeny.

In preferred embodiments of the present invention, the resultingrecombinant cells may be delivered to a patient by various methods knownwithin the art including, e.g., injection of epithelial cells (e.g.,subcutaneously), application of recombinant skin cells as a skin graftonto the patient, and intravenous injection of recombinant blood cells(e.g., hematopoietic stem or progenitor cells). The total amount ofcells that are envisioned for use depend upon the desired effect,patient state, and the like, and may be determined by one skilled withinthe art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and may bexenogeneic, heterogeneic, syngeneic, or autogeneic. Cell types include,but are not limited to, differentiated cells such as epithelial cells,endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytesand blood cells, or various stem or progenitor cells, in particularembryonic heart muscle cells, liver stem cells (International PatentPublication WO 94/08598), neural stem cells (Stemple and Anderson, 1992,Cell 71: 973-985), hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, and the like. In a preferred embodiment, the cells utilized forgene therapy are autologous to the patient.

Immunoassays

The peptides and antibodies of the present invention may be utilized inassays (e.g., immunoassays) to detect, prognose, diagnose, or monitorvarious conditions, diseases, and disorders characterized by aberrantlevels of JNK, or a JNK inhibitor peptide, or monitor the treatmentthereof. An “aberrant level” means an increased or decreased level in asample relative to that present in an analogous sample from anunaffected part of the body, or from a subject not having the disorder.The immunoassay may be performed by a method comprising contacting asample derived from a patient with an antibody under conditions suchthat immunospecific-binding may occur, and subsequently detecting ormeasuring the amount of any immunospecific-binding by the antibody. In aspecific embodiment, an antibody specific for a JNK inhibitor peptidemay be used to analyze a tissue or serum sample from a patient for thepresence of JNK or a JNK inhibitor peptide; wherein an aberrant level ofJNK or a JNK inhibitor peptide is indicative of a diseased condition.The immunoassays that may be utilized include, but are not limited to,competitive and non-competitive assay systems using techniques such asWestern Blots, radioimmunoassays (RIA), enzyme linked immunosorbentassay (ELISA), “sandwich” immunoassays, immunoprecipitation assays,precipitin reactions, gel diffusion precipitin reactions,immunodiffusion assays, agglutination assays, fluorescent immunoassays,complement-fixation assays, immunoradiometric assays, and protein-Aimmunoassays, etc.

Kits

The present invention additionally provides kits for diagnostic ortherapeutic use that include one or more containers containing ananti-JNK inhibitor peptide antibody and, optionally, a labeled bindingpartner to the antibody. The label incorporated into the antibody mayinclude, but is not limited to, a chemiluminescent, enzymatic,fluorescent, colorimetric or radioactive moiety. In another specificembodiment, kits for diagnostic use that are comprised of one or morecontainers containing modified or unmodified nucleic acids that encode,or alternatively, that are the complement to, a JNK inhibitor peptideand, optionally, a labeled binding partner to the nucleic acids, arealso provided. In an alternative specific embodiment, the kit maycomprise, in one or more containers, a pair of oligonucleotide primers(e.g., each 6-30 nucleotides in length) that are capable of acting asamplification primers for polymerase chain reaction (PCR; see, e.g.,Innis, et al., 1990. PCR PROTOCOLS, Academic Press, Inc., San Diego,Calif.), ligase chain reaction, cyclic probe reaction, and the like, orother methods known within the art. The kit may, optionally, furthercomprise a predetermined amount of a purified JNK inhibitor peptide, ornucleic acids thereof, for use as a diagnostic, standard, or control inthe assays.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications fall within the scope of the appendedclaims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entirety.

SPECIFIC EXAMPLES Example 1 Identification of JNK Inhibitor Peptides

Amino acid sequences important for efficient interaction with JNK wereidentified by sequence alignments between known JBDs. Sequencecomparison between the JBDs of IB1 [SEQ ID NO:17], IB2 [SEQ ID NO:18],c-Jun [SEQ ID NO:19] and ATF2 [SEQ ID NO:20] defined a weakly conserved8 amino acid sequence (FIG. 1A). Since the JBDs of IB1 and IB2 areapproximately 100 fold as efficient as c-Jun or ATF2 in binding MNK(Dickens et al. Science 277: 693 (1997), it was reasoned that conservedresidues between IB1 and IB2 must be important to confer maximalbinding. The comparison between the JBDs of IB1 and IB2 defined twoblocks of seven and three amino acids that are highly conserved betweenthe two sequences. These two blocks are contained within a peptidesequence of 23 amino acids in IB1 [SEQ ID NO:1] and 21 amino acid IB2[SEQ ID NO:2]. These sequences are shown in FIG. 1B, dashes in the IB2sequence indicate a gap in the sequence in order to align the conservedresidues.

Example 2 Preparation of JNK Inhibitor Fusion Proteins

JNK inhibitor fusion proteins were synthesized by covalently linking theC-terminal end of JBD₂₃ or the 21 amino acid sequence derived from theJBD of IB2 (JBD₂₁) to a N-terminal 10 amino acid long carrier peptidederived from the HIV-TAT₄₈₋₅₇ (Vives et al., J. Biol. Chem. 272: 16010(1997)) via a spacer consisting of two proline residues. This spacer wasused to allow for maximal flexibility and prevent unwanted secondarystructural changes. As shown in FIG. 1C, these preparations weredesignated L-TAT [SEQ ID NO:7], L-TAT-IB1 [SEQ ID NO:11] and L-TAT-IB2[SEQ ID NO:12], respectively. All-D retro-inverso peptides TAT-fusionpeptides were also synthesized and were designated D-TAT [SEQ ID NO:8]and D-TAT-IB1 [SEQ ID NO:14], respectively. All D and L peptides wereproduced by classical F-mock synthesis and further analysed by MassSpectrometry. They were finally purified by HPLC. To determine theeffects of the proline spacer, two types of TAT peptide were producedone with and one without two prolines. The addition of the two prolinesdid not appear to modify the entry or the localization of the TATpeptide inside cells.

Generic peptides showing the conserved amino acid residues are given inFIG. 2. An “X” indicates any amino acid. The number of Xs in a givenpeptide is not limited to the one depicted, and may vary. See above fora more detailed description of the generic sequences.

Example 3 Inhibition of βCell Death By JBD₂₃

Effects of the 23 a.a. long JBD sequence of IB1 on JNK biologicalactivities were then studied. The 23 a.a. sequence was linked N-terminalto the Green Fluorescent Protein (GFP-JBD₂₃ construct), and the effectof this construct on pancreatic β-cell apoptosis induced by IL-1β wasevaluated. See FIG. 3. This mode of apoptosis was previously shown to beblocked by transfection with JBD₁₋₂₈₀, whereas specific inhibitors ofERK1/2 or p38 did not protect. See Ammendrup et al, supra.

Oligonucleotides corresponding to the 23 amino acid sequence (JBD₂₃;FIG. 1B) and a sequence mutated at the fully conserved regions(JBD_(23mut)) were synthesized and directionally inserted into the EcoRIand SalI sites of the pEGFP-N1 vector encoding the Green FluorescentProtein (GFP) (from Clontech). Insulin producing βTC-3 cells werecultured in RPMI 1640 medium supplemented with 10% Fetal Calf Serum, 100μg/mL Streptomycin, 100 units/mL Penicillin and 2 mM Glutamine. Insulinproducing βTC-3 cells were transfected with the indicated vectors andIL-1β (10 ng/mL) was added to the cell culture medium. The number ofapoptotic cells were counted at 48 hours after the addition of IL-1βusing an inverted fluorescence microscope. Apoptotic cells werediscriminated from normal cells by the characteristic “blebbing out” ofthe cytoplasm were counted after two days.

As indicated in FIG. 3, GFP is Green Fluorescent protein expressionvector used as a control; JBD23 is the vector expressing a chimeric GFPlinked to the 23 a.a. sequence from the JBD of IB1; JBD23Mut is the samevector as GFP-JBD23, but with a JBD mutated at four conserved residuesshown as FIG. 1B; and JBD280 is the GFP vector linked to the entire JBD(a.a. 1-280). The GFP-JBD₂₃ expressing construct prevented IL-1β inducedpancreatic β-cell apoptosis as efficiently as the entire JBD₁₋₂₈₀ (FIG.3, JBD23/IL-1 compared to JBD280/IL-1). As additional controls,sequences mutated at fully conserved IB1 residues had greatly decreasedability to prevent apoptosis (FIG. 3, JBD23Mut/IL-1).

Example 4 Cellular Import of TAT-IB1 and TAT-IB2 Peptides

The ability of the L- and D-enantiomeric forms of TAT, TAT-IB1 andTAT-IB2 peptides (“TAT-IB peptides”) to enter cells were evaluated.

L-TAT, D-TAT, L-TAT-IB1, L-TAT-IB2 and D-TAT-IB1 peptides [SEQ ID NOs:7,8, 11, 12 and 14, respectively] were labeled by N-terminal addition of aglycine residue conjugated to fluorescein. Labeled peptides (1 μM) wereadded to PTC-3 cell cultures, which were maintained as described inExample 3. At predetermined times, cells were washed with PBS and fixedfor five minutes in ice-cold methanol-acetone (1:1) before beingexamined under a fluorescence microscope. Fluorescein-labeled BSA (1 μM,12 moles/mole BSA) was used as a control. Results demonstrated that allthe above fluorescein labeled peptides had efficiently and rapidly (lessthan five minutes) entered cells once added to the culture medium.Conversely, fluorescein labeled bovine serum albumin (1 μM BSA, 12 molesfluorescein/mole BSA) did not enter the cells.

A time course study indicated that the intensity of the fluorescentsignal for the L-enantiomeric peptides decreased by 70% following a 24hours period. Little to no signal was present at 48 hours. In contrast,D-TAT and D-TAT-IB1 were extremely stable inside the cells. Fluorescentsignals from these all-D retro-inverso peptides were still very strong 1week later, and the signal was only slightly diminish at 2 weeks posttreatment.

Example 5 In Vitro Inhibition Of c-JUN, ATF2 and Elk1 Phosphorylation

The effects of the peptides on JNKs-mediate phosphorylation of theirtarget transcription factors were investigated in vitro. Recombinant andnonactivated JNK1, JNK2 and JNK3 were produced using a TRANSCRIPTION ANDTRANSLATION rabbit reticulocyte lysate kit (Promega) and used in solidphase kinase assays with c-Jun, ATF2 and Elk1, either alone or fused toglutathione-S-transferase (GST), as substrates. Dose response studieswere performed wherein L-TAT, L-TAT-IB1 or L-TAT-IB2 peptides (0-25 μM)were mixed with the recombinant JNK1, JNK2, or JNK3 kinases in reactionbuffer (20 mM Tris-acetate, 1 mM EGTA, 10 mM p-nitrophenyl-phosphate(pNPP), 5 mM sodium pyrophosphate, 10 mM p-glycerophosphate, 1 mMdithiothreitol) for 20 minutes. The kinase reactions were then initiatedby the addition of 10 mM MgCl₂ and 5 μCi ³³P-γ-dATP and 1 μg of eitherGST-Jun (a.a. 1-89), GST-AFT2 (a.a. 1-96) or GST-ELK1 (a.a. 307-428).GST-fusion proteins were purchased from Stratagene (La Jolla, Calif.).Ten μL of glutathione-agarose beads were also added to the mixture.Reaction products were then separated by SDS-PAGE on a denaturing 10%polyacrylamide gel. Gels were dried and subsequently exposed to X-rayfilms (Kodak). Nearly complete inhibition of c-Jun, ATF2 and Elk1phosphorylation by JNKs was observed at TAT-IB peptide doses as low as2.5 μM. However, a marked exception was the absence of TAT-IB inhibitionof JNK3 phosphorylation of Elk1. Overall, the TAT-IB1 peptide appearedslightly superior to TAT-IB2 in inhibiting JNK family phosphorylation oftheir target transcription factors. (See, FIG. 4A).

The ability of D-TAT, D-TAT-IB1 and L-TAT-IB1 peptides (0-250 μM dosagestudy) to inhibit GST-Jun (a.a. 1-73) phosphorylation by recombinantJNK1, JNK2, and JNK3 by were analyzed as described above. Overall,D-TAT-IB1 peptide decreased JNK-mediated phosphorylation of c-Jun, butat levels approximately 10-20 fold less efficiently than L-TAT-IB1.(See, FIG. 4B).

Example 6 Inhibition of c-JUN Phosphorylation by Activated JNKS

The effects of the L-TAT, L-TAT-IB1 or L-TAT-IB2 peptides on JNKsactivated by stressful stimuli were evaluated using GST-Jun to pull downJNKs from UV-light irradiated HeLa cells or IL-1β treated βTC cells. βTCcells were cultured as described above. HeLa cells were cultured in DMEMmedium supplemented with 10% Fetal Calf Serum, 100 μg/mL Streptomycin,100 units/ml Penicillin and 2 mM Glutamine. One hour prior to being usedfor cell extract preparation, PTC cells were activated with IL-1β asdescribed above, whereas HeLa cells were activated by UV-light (20J/m²). Cell extracts were prepared from control, UV-light irradiatedHeLa cells and IL-1β treated βTC-3 cells by scraping the cell culturesin lysis buffer (20 mM Tris-acetate, 1 mM EGTA, 1% Triton X-100, 10 mMp-nitrophenyl-phosphate, 5 mM sodium pyrophosphate, 10 mMβ-glycerophosphate, 1 mM dithiothretiol). Debris was removed bycentrifugation for five minutes at 15,000 rpm in an SS-34 Beckman rotor.One-hundred μg extracts were incubated for one hour at room temperaturewith one μg GST-jun (amino acids 1-89) and 10 μL of glutathione-agarosebeads (Sigma). Following four washes with the scraping buffer, the beadswere resuspended in the same buffer supplemented with L-TAT, L-TAT-IB1or L-TAT-IB2 peptides (25 μM) for 20 minutes. Kinase reactions were theninitiated by the addition of 10 mM MgCl₂ and 5 μCi ³³P-γ-dATP andincubated for 30 minutes at 30° C. Reaction products were then separatedby SDS-PAGE on a denaturing 10% polyacrylamide gel. Gels were dried andsubsequently exposed to X-ray films (Kodak). The TAT-IB peptidesefficiently prevented phosphorylation of c-Jun by activated JNKs inthese experiments. (See, FIG. 6).

Example 7 In vivo Inhibition of c-JUN Phosphorylation by TAT-IB Peptides

To determine whether the cell-permeable peptides could block JNKsignaling in vivo, we used a heterologous GAL4 system. HeLa cells,cultured as described above, were co-transfected with the 5×GAL-LUCreporter vector together with the GAL-Jun expression construct(Stratagene) comprising the activation domain of c-Jun (amino acids1-89) linked to the GAL4 DNA-binding domain. Activation of JNK wasachieved by the co-transfection of vectors expressing the directlyupstream kinases MKK4 and MKK7 (See, Whitmarsh et al., Science 285: 1573(1999)). Briefly, 3×10⁵ cells were trasfected with the plasmids in3.5-cm dishes using DOTAP (Boehringer Mannheim) following instructionsfrom the manufacture. For experiments involving GAL-Jun, 20 ng of theplasmid was transfected with 1 μg of the reporter plasmid pFR-Luc(Stratagene) and 0.5 μg of either MKK4 or MKK7 expressing plasmids.Three hours following transfection, cell media were changed and TAT,TAT-IB1, and TAT-IB2 peptides (1 μM) were added. The luciferaseactivities were measured 16 hours later using the “Dual Reporter System”from Promega after normalization to protein content. As shown in FIG. 5,addition of both the TAT-IB1 and TAT-IB2 peptides blocked activation ofc-Jun following MKK4 and MKK7 mediated activation of JNK. Because HeLacells express both JNK1 and JNK2 isoforms but not JNK3, we transfectedcells with JNK3. Again, the two TAT-IB peptides inhibited JNK2 mediatedactivation of c-Jun.

Example 8 Inhibition of IL-1β Induced Pancreatic β-Cell Death by TAT-IBPeptides

We investigated the effects of the L-TAT-IB peptides on the promotion ofpancreatic β-cell apoptosis elicited by IL-1β. βTC-3 cell cultures wereincubated for 30 minutes with 1 μM of either L-TAT-IB1 or L-TAT-IB2peptides followed by 10 ng/mL of IL-1β. A second addition of peptide (1μM) was performed 24 hours later. Apoptotic cells were counted after twodays of incubation with IL-1β using Propidium Iodide (red stained cellare dead cells) and Hoechst 33342 (blue stained cell are cells withintact plasma membrane) nuclear staining. As shown in FIG. 5, additionof the TAT-IB peptides inhibited IL-1β-induced apoptosis of β TC-3 cellscultured in the presence of IL-1β for two days.

Long term inhibition of IL-1β induced cells death was examined bytreating βTC-3 cells as described above, except that incubation of thecells with the peptides and IL-1β was sustained for 12 days. Additionalpeptides (1 μM) were added each day and additional IL-1β (10 ng/mL) wasadded every 2 days. The TAT-IB1 peptide confers strong protectionagainst apoptosis in these conditions. Taken together, these experimentsestablish that TAT-IB peptides are biologically active molecules able toprevent the effects of JNK signaling on cell fate.

Example 9 Synthesis of an All-D-Retro-Inverso Peptides

Peptides of the invention may be all-D amino acid peptides synthesizedin reverse to prevent natural proteolysis (i.e., all-D-retro-inversopeptides). An all-D retro-inverso peptide of the invention would providea peptide with functional properties similar to the native peptide,wherein the side groups of the component amino acids would correspond tothe native peptide alignment, but would retain a protease resistantbackbone.

Retro-inverso peptides of the invention are analogs synthesized usingD-amino acids by attaching the amino acids in a peptide chain such thatthe sequence of amino acids in the retro-inverso peptide analog isexactly opposite of that in the selected peptide which serves as themodel. To illustrate, if the naturally occurring TAT protein (formed ofL-amino acids) has the sequence GRKKRRQRRR [SEQ ID NO:7], theretro-inverso peptide analog of this peptide (formed of D-amino acids)would have the sequence RRRQRRKKRG [SEQ ID NO:8]. The procedures forsynthesizing a chain of D-amino acids to form the retro-inverso peptidesare known in the art. See, e.g., Jameson et al., Nature, 368, 744-746(1994); Brady et al., Nature, 368, 692-693 (1994)); Guichard et al., J.Med. Chem. 39, 2030-2039 (1996). Specifically, the retro-peptides wereproduced by classical F-mock synthesis and further analysed by MassSpectrometry. They were finally purified by HPLC.

Since an inherent problem with native peptides is degradation by naturalproteases and inherent immunogenicity, the heterobivalent orheteromultivalent compounds of this invention will be prepared toinclude the “retro-inverso isomer” of the desired peptide. Protectingthe peptide from natural proteolysis should therefore increase theeffectiveness of the specific heterobivalent or heteromultivalentcompound, both by prolonging half-life and decreasing the extent of theimmune response aimed at actively destroying the peptides.

Example 10 Long Term Biological Activity of All-D-Retro-Inverso IBPeptides

Long term biological activity is predicted for the D-TAT-IBretro-inverso containing peptide heteroconjugate when compared to thenative L-amino acid analog owing to protection of the D-TAT-IB peptidefrom degradation by native proteases, as shown in Example 5.

Inhibition of IL-1β induced pancreatic β-cell death by the D-TAT-1βpeptide was analyzed. As shown in FIG. 10, βTC-3 cells were incubated asdescribed above for 30 minutes with one single addition of the indicatedpeptides (1 μM), then IL-1β (10 ng/ml) was added. Apoptotic cells werethen counted after two days of incubation with IL-1β by use of PropidiumIodide and Hoechst 33342 nuclear staining. A minimum of 1,000 cells werecounted for each experiment. Standard Error of the Means (SEM) areindicated, n=5. The D-TAT-IB1 peptide decreased IL-1 induced apoptosisto a similar extent as L-TAT-IB peptides (compare FIG. 5 and FIG. 10).

Long term inhibition of IL-1β induced cell-death by the D-TAT-IB1peptide was also analyzed. βTC-3 cells were incubated as above for 30minutes with one single addition of the indicated peptides (1 μM), thenIL-1β (10 ng/ml) was added, followed by addition of the cytokine everytwo days. Apoptotic cells were then counted after 15 days of incubationwith IL-1β by use of Propidium Iodide and Hoechst 33342 nuclearstaining. Note that one single addition of the L-TAT-IB1 peptide doesnot confer long-term protection. A minimum of 1,000 cells were countedfor each experiment. Standard Error of the Means (SEM) are indicated,n=5. Results are shown in FIG. 9. D-TAT-IB1, but not L-TAT-IB1, was ableto confer long term (15 day) protection.

Example 11 Inhibition of Irradiation Induced Pancreatic β-Cell Death byTAT-IB Peptides

JNK is also activated by ionizing radiation. To determine whether TAT-IBpeptides would provide protection against radiation-induced JNK damage,“WiDr” cells were irradiated (30Gy) in presence or absence of D-TAT,L-TAT-IB1 or D-TAT-IB1 peptides (1 μM added 30 minutes beforeirradiation), as indicated in FIG. 10. Control cells (CTRL) were notirradiated. Cells were analyzed 48 hours later by mean of PI and Hoechst33342 staining, as described above. n=3, SEM are indicated. L-TAT-IB1and D-TAT-1β peptides were both able to prevent irradiation inducedapoptosis in this human colon cancer cell line.

Example 12 Radioprotection to Ionizing Radiation by TAT-IB Peptides

To determine the radioprotective effects of the TAT-IB peptides, C57B1/6 mice (2 to 3 months old) were irradiated with a Phillips RT 250R-ray at a dose rate of 0.74 Gy/min (17 mA, 0.5 mm Cu filter). Thirtyminutes prior to irradiation, the animals were injected i.p. with eitherthe TAT, L-TAT-IB1 and D-TAT-IB1 peptides (30 μl of a 1 mM solution).Briefly, mice were irradiated as follows: mice were placed in smallplastic boxes with the head lying outside the box. The animals wereplaced on their back under the irradiator, and their neck fixed in asmall plastic tunnel to maintain their head in a correct position. Thebody was protected with lead. Prior to irradiation mice were maintainedon standard pellet mouse chow, however post irradiation mice were fedwith a semi-liquid food that was renewed each day.

The reaction of the lip mucosa was then scored by 2 independentobservers according to the scoring system developed by Parkins et al.(Parkins et al, Radiotherapy & Oncology, 1: 165-173, 1983), in which theerythema status as well as the presence of edema, desquamation andexudation was quoted. Additionally, animals were weighed before eachrecording of their erythema/edema status.

FIG. 12A: illustrated the weight of the mice following irradiation.Values are reported to the initial weight of the mice that was set to100. CTRL: control mice injected with 30 μl of a saline solution. n=2for each values reported, S.D. are indicated. x values are days

FIG. 12B is illustrative of the erythema/edema scoring followingirradiation. The edema and erythema status of the ventral lip of thesame mice as in FIG. 12A was quantified. n=2 for each value reported. xvalues are days

The results of these experiments indicate that the TAT-IB Peptides canprotect against weight loss and erythema/edema associated with ionizingradiation.

Example 13 Suppression of JNK Transcription Factors by L-TAT-IB1Peptides

Gel retardation assays were carried out with an AP-1 doubled labeledprobe (5′-CGC TTG ATG AGT CAG CCG GAA-3′. HeLa cell nuclear extractsthat were treated or not for one hour with 5 ng/ml TNF-α, as indicated.TAT and L-TAT-IB1 peptides were added 30 minutes before TNF-α. Only thepart of the gel with the specific AP-1 DNA complex (as demonstrated bycompetition experiments with non-labeled specific and non-specificcompetitors) is shown. L-TAT-IB1 peptides decrease the formation of theAP-1 DNA binding complex in the presence of TNF-α. (See, FIG. 11).

EQUIVALENTS

From the foregoing detailed description of the specific embodiments ofthe invention, it should be apparent that unique cell-permeablebioactive peptides have been described. Although particular embodimentshave been disclosed herein in detail, this has been done by way ofexample for purposes of illustration only, and is not intended to belimiting with respect to the scope of the appended claims which follow.In particular, it is contemplated by the inventor that varioussubstitutions, alterations, and modifications may be made to theinvention without departing from the spirit and scope of the inventionas defined by the claims.

1.-37. (canceled)
 38. A D-enantiomeric peptide comprising the amino acidsequence of SEQ ID NO: 8, wherein said peptide facilitates transportacross a biological membrane.
 39. A transport protein comprising aD-enantiomeric peptide, said D-enantiomeric peptide consisting of theamino acid sequence of SEQ ID NO:
 8. 40. A polypeptide comprising thepeptide of claim 38 and a protein covalently bound to said peptide. 41.A transport protein consisting of a D-enantiomeric peptide, saidD-enantiomeric peptide consisting of the amino acid sequence of SEQ IDNO: 8.